This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2001-242634, filed Aug. 9, 2001, the entire contents of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates to a laser microscope which condenses a laser beam onto a specimen and detects the light obtained from the specimen.
2. Description of the Related Art
For fluorescent observation of a specimen such as a living tissue by laser microscope, a specimen is at present colored with a plurality of fluorescent reagents. In other words, a plurality of fluorescent reagents is used to color one specimen. A plurality of different wavelength laser beams is irradiated onto the specimen in multiple fluorescence observation. The form and function of a living body cell can be analyzed using this method.
For instance, when an excited light (a laser beam) with a single wavelength (hereinafter referred to as 1-wavelength) is irradiated onto a specimen. There is a reagent which generates 1-wavelength excited 2-wavelength fluorescence, which fluorescence in two wavelength on the specimen (hereinafter referred to as 2-wavelengths). There is another reagent which evokes energy transfer (Fluorescence Resonance Energy Transfer) which excites another fluorescence coloring material by the energy of one excited fluorescence coloring material. It is practical to obtain the fluorescence quantity ratio of such fluorescence by observing a 2-wavelength fluorescence with respect to a 1-wavelength excited beam.
As a reagent which generates 1-wavelength excited 2-wavelength fluorescence, SNARF-1 for example is known. SNARF-1 is used for measurement of pH. Recently, a probe called Cameleon (trademark) has been used for measurement of calcium ion density. This utilizes the energy transfer of two fluorescence proteins CFP and YFP. That is, CFP is excited by a laser beam with a predetermined wavelength, and YFP is excited by the energy of this excited CFP. And, the fluorescence quantity ratio of CFP wavelength and YFP wavelength is measured.
Further, a laser microscope utilizing multi-photon excitation evoked by a laser beam emitted from an ultra-short pulse laser source has been used. An ultra-short pulse laser causes multi-photon excitation, excites a fluorescence reagent for generating ultraviolet-rays in near infrared wavelength, and resultantly generates fluorescence. This type of microscope is capable of observing a specimen deeply with little influence to the specimen.
However, it is disadvantageous to detect fluorescence by using the optical system similar to that of an ordinary confocal microscope. Use of a number of lenses and mirrors increases transmission loss and decreases the intensity of the detectable fluorescence.
U.S. patent application Ser. No. 09/525,165 (now U.S. Pat. No. 6,437,913 issued Aug. 20, 2002) discloses a laser microscope technology, which separates fluorescence without returning it to the scanning optical system and confocal pinhole. This technology proposed a microscope to effectively detect fluorescence by making the optical path short with minimized loss.
A microscope using this technology is provided with a laser light source and photodetector for confocal detection, and an ultra-short pulse laser light source and photodetector to emit a laser beam to evoke multi-photon excitation. A beam splitter for confocal detection and a beam splitter for detection of the fluorescence generated by multi-photon excitation are also provided between the objective lens and the scanning optical system. These beam splitters are switched for confocal detection of fluorescence and detection of fluorescence generated by multi-photon excitation.
Further, a laser beam to be emitted and beam splitters must be switched for detection of fluorescence, in the above U.S. patent application Ser. No. 09/525,165 (now U.S. Pat. No. 6,437,913). This makes it difficult to simultaneously observe and measure a plurality of forms and functions of a living body tissue.
The present invention is to provide a laser microscope which can simultaneously detect a plurality of lights obtained from a specimen when a laser beam is irradiated onto the specimen.
According to an aspect of the invention, there is provided a laser microscope for observing a specimen, comprising:
a laser light source to radiate a laser beam;
a scanning optical system to scan the laser beam radiated from the laser light source in at least one direction;
an objective lens to condense the laser beam scanned by the scanning optical system onto a specimen, and to receive from the specimen the light generated by a linear phenomenon and the light generated by a non-linear phenomenon evoked by condensation and the reflected light of the laser beam;
a first beam splitter which is arranged between the objective lens and the scanning optical system, and selectively separates the laser beam, the light obtained from the specimen by the linear phenomenon and the reflected light, from the light obtained from the specimen by the non-linear phenomenon;
a first photodetector to detect the light generated by the non-linear phenomenon and separated by the first beam splitter;
a second beam splitter which is arranged between the laser light source and the scanning optical system, and separates the laser beam from the light obtained from the specimen by the linear phenomenon and the reflected light; and,
a second photodetector which has a pinhole arranged at a position conjugative to the focal point of the objective lens, and detects the light passing the pinhole among the light generated by the linear phenomenon and the reflected light separated by the second beam splitter.
Additional advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.